Tania Pilli

The Effects of Recombinant TSH on Bone Turnover Markers and Serum Osteoprotegerin and RANKL Levels

OBJECTIVE Recently it was found that thyrotropin (TSH) receptors are present both in osteoclast and osteoblast and that TSH can modulate bone remodeling independent of thyroid hormones. The aim of this study was, firstly, to evaluate the effects of acute administration of TSH on bone remodeling markers both in men and in women and, secondly, to evaluate if these effects are mediated by variations in serum osteoprotegerin (OPG) and receptor activator of nuclear factor-KB ligand (RANKL). DESIGN We studied 30 thyroidectomized patients (10 premenopausal and 10 postmenopausal women, 10 men) affected by thyroid carcinoma on l-thyroxine therapy. Eighty age- and sex-matched subjects were used as controls. A blood sample was drawn from each patient at baseline and 3 and 5 days after recombinant human TSH (rhTSH) administration, in preparation for (131)I whole body scan, to assess serum bone markers and serum OPG and RANKL levels. MAIN OUTCOME At baseline, postmenopausal women and men had significantly higher values of bone turnover markers and serum OPG compared to control subjects. In all thyroidectomized patients serum RANKL was lower than in controls. After rhTSH administration, serum N-terminal propeptide of type-I procollagen (PINP), a marker of bone formation, increased significantly in postmenopausal women, while serum RANKL significantly increased after 3 days in postmenopausal patients and men returning to baseline values at day 5. Serum OPG levels did not change significantly. CONCLUSIONS The low serum TSH observed in thyroidectomized patients on l-thyroxine therapy is associated with an increase of bone turnover in postmenopausal women and men that is associated with an increase of OPG and a decrease of serum RANKL levels. The acute TSH administration results in an increase of PINP, an index of osteoblastic activity, associated with an increase of serum RANKL. The lack of this response in premenopausal women suggests an influence of estrogen status on bone reactivity to TSH.

MADD Knock-Down Enhances Doxorubicin and TRAIL Induced Apoptosis in Breast Cancer Cells

The Map kinase Activating Death Domain containing protein (MADD) isoform of the IG20 gene is over-expressed in different types of cancer tissues and cell lines and it functions as a negative regulator of apoptosis. Therefore, we speculated that MADD might be over-expressed in human breast cancer tissues and that MADD knock-down might synergize with chemotherapeutic or TRAIL-induced apoptosis of breast cancer cells. Analyses of breast tissue microarrays revealed over-expression of MADD in ductal and invasive carcinomas relative to benign tissues. MADD knockdown resulted in enhanced spontaneous apoptosis in human breast cancer cell lines. Moreover, MADD knockdown followed by treatment with TRAIL or doxorubicin resulted in increased cell death compared to either treatment alone. Enhanced cell death was found to be secondary to increased caspase-8 activation. These data indicate that strategies to decrease MADD expression or function in breast cancer may be utilized to increase tumor cell sensitivity to TRAIL and doxorubicin induced apoptosis.

IFNγ-Inducible Chemokines Decrease upon Selenomethionine Supplementation in Women with Euthyroid Autoimmune Thyroiditis: Comparison between Two Doses of Selenomethionine (80 or 160 μg) versus Placebo

Background: Several studies have suggested that selenium may influence the natural history of autoimmune thyroiditis (AIT). Recently, IFNγ-inducible chemokines (CXCL-9, -10 and -11) were shown to be elevated in AIT patients. Objective: This prospective, randomized, controlled study was conducted to evaluate the effect of two doses of selenomethionine (Semet; 80 or 160 µg/day) versus placebo in euthyroid women with AIT, in terms of reduction of anti-thyroid antibodies, CXCL-9, -10 and -11 and improvement of thyroid echogenicity, over 12 months. Patients and Methods: Sixty patients, aged 21-65 years, were equally randomized into 3 groups: placebo, 80 µg/day of Semet (80-Semet) or 160 µg/day of Semet (160-Semet). Results: Anti-thyroperoxidase antibody (TPOAb) levels remained unaffected by Semet supplementation; anti-thyroglobulin antibody levels showed a significant reduction in the 160-Semet and the placebo group at 12 months. No significant change in thyroid echogenicity, thyroid volume and quality of life was observed within and between the groups. Subclinical hypothyroidism was diagnosed in 2 patients of the placebo group versus 1 patient in each Semet group. Serum CXCL-9 and -10 were significantly reduced in both Semet groups at 6 and 12 months, while they remained unchanged or increased in the placebo group. CXCL-11, TNFα and IFNγ showed a transient decrease at 6 months in both Semet groups but returned nearly to the basal levels at 12 months. Conclusions: Semet supplementation had no positive effect on thyroid echogenicity or TPOAb in our patients. However, we observed a Semet-dependent downregulation of the IFNγ-inducible chemokines, especially CXCL-9 and -10, which may serve as helpful biomarkers in future selenium supplementation trials.

Down-Modulation of Expression, or Dephosphorylation, of IG20/MADD in Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Resistant Thyroid Cancer Cells Makes Them Susceptible to Treatment with This Ligand

BACKGROUND The IG20/MADD gene is overexpressed in thyroid cancer tissues and cell lines, and can contribute to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance. The ability of the MADD protein to resist TRAIL-induced apoptosis is dependent upon its phosphorylation by Akt. Interestingly, while TRAIL induces a significant reduction in the levels of phospho-Akt (pAkt) and phospho-MADD (pMADD) in TRAIL-sensitive cells, it fails to do so in TRAIL-resistant cells. In this study, we investigated if MADD phosphorylation by Akt was contributing to TRAIL resistance in thyroid cancer cells. METHODS We determined the susceptibility of different thyroid cancer cell lines to TRAIL-induced apoptosis by fluorescence-activated cell sorting (FACS) analysis. We tested for various TRAIL resistance factors by FACS analyses or for IG20/MADD expression by quantitative reverse transcription-polymerase chain reaction. We determined the levels of pAkt and pMADD upon TRAIL treatment in thyroid cancer cells by Western blotting. We tested if down-modulation of IG20/MADD gene expression using shRNA or phosphorylation using a dominant negative Akt (DN-Akt) or pretreatment with LY294002, a PI3 kinase inhibitor, could help overcome TRAIL resistance. RESULT BCPAP and TPC1 cells were susceptible, while KTC1 and FTC133 cells were resistant, to TRAIL-induced apoptosis. The differential susceptibility to TRAIL was not related to the levels of expression of death receptors, decoy receptors, or TRAIL. KTC1 and FTC133 cells showed higher levels of IG20/MADD expression relative to BCPAP and TPC1, and were rendered susceptible to TRAIL treatment upon IG20/MADD knockdown. Interestingly, upon TRAIL treatment, the pAkt and pMADD levels were reduced in TRAIL-sensitive BCPAP and TPC1 cells, while they remained unchanged in the resistant KTC1 and FTC133 cells. While expression of a constitutively active Akt in BCPAP and TPC1 cells rendered them resistant to TRAIL, pretreating KTC1 and FTC133 cells with LY294002 rendered them TRAIL-sensitive. Moreover, expression of a DN-Akt in KTC1 and FTC133 cells reduced the levels of pAkt and pMADD and sensitized them to TRAIL-induced apoptosis. CONCLUSION Our results show that pMADD is an important TRAIL resistance factor in certain thyroid cancer cells and suggest that down-modulation of either IG20/MADD expression or phosphorylation can render TRAIL-resistant thyroid cancer cells sensitive to TRAIL.

Molecular Signature of Indeterminate Thyroid Lesions: Current Methods to Improve Fine Needle Aspiration Cytology (FNAC) Diagnosis

Fine needle aspiration cytology (FNAC) represents the gold standard for determining the nature of thyroid nodules. It is a reliable method with good sensitivity and specificity. However, indeterminate lesions remain a diagnostic challenge and researchers have contributed molecular markers to search for in cytological material to refine FNAC diagnosis and avoid unnecessary surgeries. Nowadays, several “home-made” methods as well as commercial tests are available to investigate the molecular signature of an aspirate. Moreover, other markers (i.e., microRNA, and circulating tumor cells) have been proposed to discriminate benign from malignant thyroid lesions. Here, we review the literature and provide data from our laboratory on mutational analysis of FNAC material and circulating microRNA expression obtained in the last 6 years.

Prospective Validation of ATA and ETA Sonographic Pattern Risk of Thyroid Nodules Selected for FNAC

Context Recently, the American Thyroid Association (ATA) and the European Thyroid Association (ETA) have proposed that thyroid ultrasound (US) should be used to stratify the risk of malignancy in thyroid nodules and to aid decision-making about whether fine-needle aspiration cytology (FNAC) is indicated. Objective To validate and to compare the ATA and ETA US risk stratification systems of thyroid nodules in a prospective series of thyroid nodules submitted to FNAC. Setting We prospectively evaluated 432 thyroid nodules selected for FNAC from 340 patients. Cytology reports were based on the five categories according to the criteria of the British Thyroid Association. Results The proportion of Thy2 nodules decreased significantly, whereas the proportion of Thy4/Thy5 nodules significantly increased with increasing US risk class (P < 0.0001). The ability to identify benign and malignant nodules was similar between ATA and ETA systems. According to ATA and ETA US risk stratification systems, 23.7% and 56.0% nodules did not meet the criteria for FNAC, respectively. Considering only categories at lower risk of malignancy, the cumulative malignancy rate in these nodules was 1.2% for ATA and 1.7% for ETA US risk stratification systems. Conclusions ETA and ATA US risk stratification systems provide effective malignancy risk stratification for thyroid nodules. In clinical practice, using this approach, we should be able to reduce the number of unnecessary FNAC without losing clinically relevant thyroid cancer.

MicroRNA expression profile of thyroid nodules in fine-needle aspiration cytology: a confirmatory series

IntroductionMiRNAs are small endogenous non-coding RNAs implicated with gene expression regulation. Changes in miRNA levels have been reported in thyroid cancer. Fine-needle aspiration cytology (FNAC) is the most reliable tool for differential diagnosis of thyroid nodules.MethodsWe have analyzed 174 FNAC from 168 patients with thyroid nodules for expression levels of 11 miRNAs (miRNA197; -187; -181b-3p; -181b-5p; -224; -181a; 146b; -221; -222; -155 and miRNA183) known to be up-regulated in cancer tissues compared to benign lesions. Expression of miRNAs was analyzed in FNA samples calculating the fold change of miRNA expression relative to normal thyroid tissue after normalization to an endogenous control.ResultsIn FNAC, miRNA expression was confirmed to be higher in malignant or suspicious for malignancy nodules compared to benign, only for miRNA146b, -222 and -221 (fold change expression ≥ 5).ConclusionIn this study, we confirmed that a limited set of miRNAs can be used for the differential diagnosis of thyroid nodules.

Clinical significance of type 2 iodothyronine deiodinase polymorphism

ABSTRACT Introduction: Biological activity of thyroid hormones (TH) is regulated by enzymes known as deiodinases. The most important is represented by the type 2 deiodinase (D2), which is the main T4-activating enzyme, ubiquitous in human tissues and therefore essential in many metabolic processes. A single nucleotide polymorphism (SPN) of D2, known as Thr92Ala (rs225014), has been reported in the general population while other polymorphisms are less frequently described. Areas covered: Several authors investigated the potential metabolic effect of these polymorphisms in the general population and in specific groups of patients. Thr92Ala polymorphism was mainly studied in patients with autoimmune or surgical hypothyroidism and in patients with physical/psychological disorders that could be related to an overt hypothyroidism. Susceptibility to develop more severe type 2 diabetes or insulin resistance has also been evaluated. Commentary: There is an increasing evidence that the presence of D2 polymorphisms may play a pivotal role in a better definition and customized therapeutic approach of patients with hypothyroidism and/or type 2 diabetes, suggesting that these patients should be screened for D2 polymorphisms. Nevertheless, further research should be performed in order to clarify the association between D2 polymorphisms, metabolic alterations and clinical conditions of the carrier patients.